Plasma membrane calcium ATPases (PMCAs) pump Ca2+ out of all animal cells. As components of the Ca2+ signaling "toolbox", the PMCAs are localized in distinct plasma membrane domains. The importance of proper membrane targeting is illustrated by diseases characterized by the absence of specific PMCA isoforms in the membrane, such as deafness caused by a lack of PMCA2 in cochlear hair cell stereocilia. The long-term goal is to understand the mechanism and functional impact of specific membrane targeting of the PMCAs. The specific aims are (1) to identify the apical targeting elements in PMCA2 splice variants;(2) to determine if and how PDZ protein interactions stabilize PMCA2 in the apical membrane;(3) to determine if specific localization of PMCA2 splice variants alters trans-epithelial Ca2+ flux and global Ca2+ signaling in polarized epithelial cells;and (4) to determine the subcellular distribution and identify neuronal targeting elements of PMCA2 variants in hippocampal neurons. The studies will involve confocal fluorescence microscopy and two-hybrid interaction analyses to identify specific targeting elements of PMCA2 splice variants. Fluorescence recovery after photobleaching and half-life studies will be employed to analyze the membrane dynamics of PMCA2 isoforms, and light and electron microscopy will be used to determine the localization of these pumps in adult rat hippocampus and cultured neurons. Functional studies will involve trans-epithelial Ca2+ flux measurements and ratiometric Ca2+ imaging in polarized MDCK kidney cells. This work will explore the novel concept that the physiological role of the PMCAs is tightly linked to their precise localization in the membrane. Relevance to public health: PMCA2 is abundant in neurons and is specifically localized within these cells. Although intracellular Ca2+ movements are an essential part of neurotransduction, the relative roles of the channels and pumps which control Ca2+ movements are not well understood. These studies will help us understand the mechanisms by which PMCA2 contributes to control of Ca2+. Understanding these mechanisms will help us understand and fight diseases caused by defects in local calcium regulation, such as hearing loss and neuronal degeneration in aging.